The submerged-culture fermentation of Coriolus versicolor in milk permeate based media and the characterisation ot its bioactive polysaccharides : a thesis presented in fulfilment of the requirements for the degree of Master in Food Technology

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Massey University
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The protein-bound polysaccharides of Coriolus versicolor (CPS) are known to improve human immune functions against cancers and various infectious diseases. With the increasing concern for longer lifespan and better quality of life, CPS may find new application in the food industry as a novel nutriceutical. The commercial therapeutic CPS are the intracellular polysaccharides of Coriolus versicolor (IPS) produced either through the submerged-culture fermentation or from the extract of the fruit body of Coriolus versicolor. The objective of this study was to characterise the IPS and EPS (the extracellular polysaccharides) produced by C. versicolor through submerged-culture fermentation that was based on cost-effective milk permeate (MP) as a medium component. Ten C. versicolor species were screened and the strain Wr-74 was found to be the most comparable to the patented ATCC-20545 strain in terms of EPS/EPS production, morphology of the fruit bodies and weight-average molecular weight distributions of EPS/IPS. In addition, in vitro physiological activities of EPS, IPS and mushroom extracts on cytokine production were investigated using murine splenocytes. The growth medium was optimised using MP as a base component, indicating the 50%MP-YEG medium was comparable to YEG medium in terms of EPS/IPS levels. When the lactose in MP was hydrolysed (HMP) by 0.04% (v/v) Maxilact® lactase, a 40-60% increase in EPS/IPS levels was observed in the 50%HMP-YEG medium compared to 50%MP-YEG medium. Approximately twice the amounts of EPS (3.2 mg/mL) and IPS (0.3 mg/mL) were obtained in the 100%HMP-YEG medium compared to the 50%HMP-YEG medium. Different nitrogen sources were screened and yeast extract supplied by DIFCO was found to be the most suitable for EPS/IPS production. Though glucose was the main carbon source consumed, excess amount of glucose would retard EPS/IPS production. The optimum carbon to nitrogen ratio (C/N) ratio was approximately 70. Submerged-culture fermentation was conducted using a modified impeller-assistant airlift fermenter. This fermenter was equipped with an inner draught tube and helical impeller to produce an efficient circulation of broth and dissolved oxygen. Wr-74 produced higher levels of EPS than ATCC-20545 in the fermenter. An approximately 74% increase in EPS level was obtained in the 50%HMP-YEG medium compared to 50%MP-YEG medium. The EPS levels were close to those obtained in the shake flasks (~2.5 mg/mL). The effects of antifoaming agents and salt addition were also investigated in the fermenter. Agitation with the helical impeller appeared to improve the EPS production, but resulted in lower biomass levels. Studies on the EPS/IPS compositions using HPLC indicated that the EPS from both strains only contained glucose. However, the IPS probably contained galactose, mannose or/and xylose in addition to glucose. Ratios of polysaccharide to protein were approximately 15:1 and 25:1 for EPS from ATCC-20545 and Wr-74, respectively. The EPS from both strains contained about 80% of pure polysaccharides. Approximately 82% of IPS and 32% of EPS from both strains could be obtained in the water-soluble form. Results of the amino acid analysis showed that the IPS from both strains contained higher levels of amino acids (16%, w/w) than the EPS (2% to 4%, w/w). The EPS and IPS from both strains had very similar molecular weight distributions. The weight-average molecular weights (Mw) of IPS from both strains were approximately ten times higher (10⁵ Da) than EPS (10⁴ Da) in the major elution range of 9.8 to 10.3 mL. The largest Mw fractions of EPS and IPS from both strains were about 10⁶ Da in the elution range of 8.2 to 8.7 mL. All samples of EPS, IPS and mushroom extracts stimulated cytokine production from murine splenocytes. Generally, lower polysaccharide levels (0.1 to 2.0 μig/mL) induced higher levels of cytokines. The mushroom extract from ATCC-20545 induced higher levels of IL-12 and IFN-γ than that from Wr-74. The IPS from the mycelia of Wr-74 induced higher IL-12 production, but lower levels of IFN-γ than that from ATCC-20545. The EPS from both strains were comparable in terms of IL-12 and IFN-γ production.
Coriolus versicolor, Polysaccharides, Fermentation